Mutations in the merlin tumor suppressor gene (NF2) cause the rare tumor disorder Neurofibromatosis Type 2 (NF2). Individuals with NF2 develop schwannomas as well as meningiomas and ependymomas throughout their lifetime. Bilateral vestibular schwannomas (VS) alone are diagnostic for NF2 and are typically not removed until hearing worsens or there is risk of brainstem compression. Resection of VS however leaves patients deaf and often with facial paralysis and balance deficits. Thus there is a great need for pharmaceuticals to slow or prevent schwannoma growth that are safe for chronic use. We adopted an unbiased chemical genomics approach to identify drug targets and compounds/drugs for NF2 schwannoma therapy. For this work we created merlin- deficient mouse and human Schwann cell lines, optimized their use in high-throughput and high-content assays, and screened commercial and custom compound libraries using robotic platforms. The results identified several PI3K and PI3K/mTOR inhibitors that selectively reduce viability of merlin-deficient mouse Schwann cells compared to wild-type Schwann cells. Kinome analysis of merlin-deficient human Schwann cells exposed to PI3K inhibitors for 24 hours revealed that cells consistently increased activity of focal adhesion kinase (FAK) and SRC kinase family members. Rewiring of kinase networks occurs in cancer cells developing drug resistance to monotherapies. Moreover, a drug combination screen revealed maximal synergy between a PI3K inhibitor and a FAK inhibitor in selectively reducing viability of a human merlin-deficient Schwann cell line. These results suggest that targeting the FAK/SRC pathway in combination with PI3K inhibitors should provide sustained inhibition of merlin-deficient Schwann cell proliferation and/or survival. In this proposal, we advance these findings by investigating the efficacy of combined PI3K pathway and FAK/SRC inhibition in cell based assays using mouse and human merlin-deficient Schwann cell lines, as well as patient-derived VS cells, and in vivo using allograft and genetic models of NF2 schwannomas. The aims of the proposal are to: 1) screen combinations of PI3K pathway and FAK/SRC inhibitors for synergistic efficacy in reducing viability of merlin- deficient Schwann cell lines; 2) pre-screen advanced drug combinations for in vivo efficacy using a rapid sciatic nerve allograft model, 3) test efficacy of one drug combination in preserving hearing and balance, and slowing schwannoma growth in a genetically engineered mouse model, and 4) assess successful drug combinations in primary human VS cells. We expect to obtain the necessary pre-clinical data to support the potential use of PI3K pathway and FAK/SRC inhibitors, alone or in combination for slowing progression of hearing loss and schwannoma growth in NF2. Moreover these studies will establish a streamlined in vitro and in vivo drug testing platform for future studies.